Polypeptide coupling compound targeting GPC3 as well as preparation method and application of polypeptide coupling compound

Abstract

The invention belongs to the field of chemical medicines, and relates to a polypeptide coupling compound targeting GPC3, the general formula of the polypeptide coupling compound is shown in the specification, in the formula, X is any polypeptide with GPC3 targeting ability; y is a medicine with chemotherapy, radiotherapy and immunotherapy functions; linker is any connexon which is intended to be coupled with the GPC3 targeting polypeptide and the drug MMAE. The polypeptide capable of being specifically combined with a liver cancer cell specific molecular marker GPC3 is coupled with a medicine MMAE with a chemotherapy-radiotherapy-immunotherapy function to obtain a target compound, namely the polypeptide coupling medicine which targets the GPC3 and has a chemotherapy-radiotherapy-immunotherapy triple-effect synergistic treatment function; in some embodiments of the compound, the molecular compound has good in-vitro biological activity, can be specifically combined with hepatoma carcinoma cells, can be efficiently internalized, completes targeted release of MMAE, can significantly induce the hepatoma carcinoma cell cycle to be detained in the most sensitive G2 / M period of radiotherapy, and highlights the application potential in precise chemoradiotherapy of hepatoma carcinoma.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a polypeptide coupling compound targeting GPC3, and a preparation method and application thereof. Background Art

[0002] Hepatocellular carcinoma (HCC) accounts for approximately 85% of liver cancers. In 2022, there will be approximately 866,000 new cases and 758,000 deaths worldwide, making it the sixth most common cancer and the third leading cause of cancer death. Treatment options include surgery, interventional therapy, ablation, targeted therapy, chemotherapy, radiotherapy, and immunotherapy. Surgery is the preferred option, but 70% of patients are diagnosed at an advanced stage. For advanced HCC, chemotherapy combined with radiotherapy is a key approach to prolonging survival. However, chemotherapy often causes systemic toxicity due to its inability to distinguish between cancer cells and normal cells. While radiotherapy can shrink tumors noninvasively, the ionizing radiation can randomly damage exposed normal tissues, limiting its efficacy. While immune checkpoint inhibitors (such as PD-1 / PD-L1) combined with anti-angiogenic drugs are first-line options, HCC tumors have low immunogenicity, an immunosuppressive microenvironment, and immune escape mechanisms, resulting in low response rates and limited survival benefits. The development of novel targeted drugs and combination therapy strategies to reverse immunosuppression and improve efficacy remains a pressing need in HCC clinical research.

[0003] Glypican-3 (GPC3) is a heparan sulfate proteoglycan anchored to the cell surface via glycosylphosphatidylinositol (GPI) and plays a key role in cell differentiation, growth, and migration. Existing studies have shown that GPC3 is highly expressed in over 70% of hepatocellular carcinoma (HCC) cells, while it is absent in normal liver tissue, making it an ideal therapeutic target for HCC. Peptide-drug conjugates (PDCs), a new generation of targeted therapy strategies following antibody-drug conjugates (ADCs), have garnered significant attention in the past decade due to their potential to overcome the limitations of ADCs. PDCs, with their small molecular weight, offer advantages such as rapid tissue distribution, deep tumor penetration, and efficient drug delivery to areas of low perfusion. Their short in vivo half-life and rapid renal clearance significantly reduce toxicity. Compared to antibodies, peptides are easier to synthesize and less expensive, making them ideal targeted delivery vehicles. PDCs consist of a targeting peptide, a payload, and a degradable linker. The targeting peptide directs the PDC to bind to tumor receptors, followed by drug release within tumor cells, inducing cell death. Since normal cells lack corresponding receptors, PDCs can enhance therapeutic efficacy while minimizing damage to healthy tissue. Based on the positive results of preclinical and clinical studies, PDC is becoming a potential tool for targeted cancer treatment. Summary of the Invention

[0004] The object of the present invention is to provide a polypeptide-coupled compound targeting GPC3.

[0005] Another object of the present invention is to provide a specific method for preparing the GPC3-targeting polypeptide coupling compound.

[0006] Another object of the present invention is to provide specific applications of the GPC3-targeting polypeptide-coupled compound.

[0007] The present invention provides a polypeptide coupling compound targeting GPC3, the general formula of which is as follows:

[0008] X——Linker——Y

[0009] Wherein, X is any polypeptide capable of targeting GPC3, including but not limited to the following polypeptide sequences: YFLTTRQ, RLNVGGTYFLTTRQ, ALLANHEELFQT, THVSPNQGGLPS, DYEMHLWWGTEL, DHLASLWWGTEL, and SNDRPPNILQKR.

[0010] Y is a drug with chemotherapy, radiotherapy, and immunotherapy functions; specifically, MMAE, a drug with "chemotherapy-radiotherapy-immunotherapy" functions, and the structural formula of the drug MMAE is as follows:

[0011]

[0012] Linker is any linker designed to couple the GPC3-targeting peptide to the drug MMAE. This includes, but is not limited to, the common Val-Cit-PABC linker, which is specifically cleavable by cathepsin B. To achieve covalent attachment of the GPC3-targeting peptide to the linker, a maleimide acetyl end is introduced at the front of the linker, and a cysteine ​​is introduced at the N-terminus of the targeting peptide. Site-specific, efficient covalent coupling is achieved via a maleimide-thiol click chemistry reaction. The specific structural formula is as follows:

[0013]

[0014] The working principle of this technical solution is to design and synthesize a variety of different types of GPC3-targeted peptide derivatives based on the peptide that can specifically bind to GPC3 as a targeted therapy carrier, and study their targeting, chemotherapy anti-tumor, combined radiotherapy anti-tumor and induction of immunogenic cell death activity and mechanism of action of liver cancer.

[0015] The specific preparation method of the above-mentioned polypeptide-coupled compound targeting GPC3 comprises the following steps:

[0016] Step S1: Preparation of the intermediate MC-Val-Cit-PABC-MMAE. The synthetic route is as follows:

[0017]

[0018] The specific preparation method is as follows: Fmoc-L-Cit-OH is used as a starting material, reacted with p-aminobenzyl alcohol under the catalytic conditions of HATU and DIPEA, and amidated to obtain compound I; compound I is de-Fmocated under the action of Et2NH to obtain compound II; compound II is reacted with Fmoc-L-Val-OH under the catalytic conditions of HATU and DIPEA to obtain compound III; compound III is de-Fmocated under the action of Et2NH to obtain compound IV; compound IV is reacted with 6-maleimidocaproic acid under the catalytic conditions of HATU and DIPEA to obtain compound V; compound V is reacted with di(p-nitrobenzyl) carbonate under the catalytic conditions of DIPEA to obtain compound VI; and compound VI is catalyzed by HOBt and pyridine to obtain the intermediate MC-Val-Cit-PABC-MMAE. Among them, HATU is 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate; DIPEA is N,N-diisopropylethylamine; HOBt is 1-hydroxybenzotriazole.

[0019] Step S2: Using the standard fluorenylmethyloxycarbonyl (Fmoc) solid-phase peptide synthesis (SPPS) method, Rink amide-MBHA resin was used as the raw material. Fmoc-protected amino acids were added. In the presence of HATU, HOBt, and DIPEA, a GPC3-targeting peptide was synthesized on solid phase. A cysteine ​​residue was introduced at the N-terminus of the GPC3-targeting peptide to synthesize peptides IX to XV. The synthetic route is as follows:

[0020]

[0021] The specific preparation method is as follows: using Rink amide-MBHA resin as a solid support, the Fmoc protecting group is deprotected with a 20% anhydrous piperidine DMF (v / v) solution, and HATU, HOBt, and DIPEA are used as coupling agents. The deprotected resin is coupled with an Fmoc-protected amino acid (5.0 equivalents) in anhydrous DMF by microwave reaction at 75°C for 5 minutes. The completion of each coupling step is confirmed by the ninhydrin method. The order of amino acid addition is the order of the peptide sequence, and the GPC3-targeting peptide with a cysteine ​​modification introduced into the N-terminus is finally obtained. Subsequently, to remove the peptide from the resin, a 95:2.5:2.5 ratio of TFA / TIPS / H2O (v / v / v) cleavage agent is added. The synthesized peptide is separated from the resin and filtered. The resulting filtrate is poured into -20°C methyl tert-butyl ether to form a large amount of white precipitate, which is collected by centrifugation. The precipitate is washed 2-3 times with cold methyl tert-butyl ether to obtain the crude peptide. Finally, peptides IX to XV were separated and purified by preparative HPLC. DMF stands for N,N-dimethylformamide, TFA stands for trifluoroacetic acid, and TIPS stands for triisopropylsilylethynyl.

[0022] Step S3: Prepare the targeted GPC3 polypeptide coupling compound, i.e., target polypeptide coupling compounds XVI to XXII. The synthetic route is as follows:

[0023]

[0024] Peptides IX to XV were mixed with MC-Val-Cit-PABC-MMAE and Et3N, respectively, reacted at room temperature for 5 hours, and purified by preparative HPLC to obtain peptide coupling compounds XVI to XXII.

[0025] The present invention provides the use of the above-mentioned polypeptide coupling compound targeting GPC3 and its salt, hydrate or pharmaceutical composition in the preparation of a drug targeting GPC3 and having "chemotherapy-radiotherapy-immunotherapy" functions.

[0026] The present invention also provides a drug for preventing, diagnosing, and / or treating diseases associated with GPC3 expression. The drug is prepared using the aforementioned GPC3-targeting polypeptide-coupled compound and other pharmaceutically acceptable carriers and / or excipients that are non-toxic and inert to humans and animals. Diseases associated with GPC3 expression include liver cancer and tumors associated with GPC3 expression.

[0027] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0028] (1) The present invention conjugates a polypeptide that can specifically bind to the liver cancer cell-specific molecular marker GPC3 with the drug MMAE that has the function of "chemotherapy-radiotherapy-immunotherapy" to obtain the target compound of the present invention, that is, a polypeptide-conjugated drug that targets GPC3 and has the triple synergistic therapeutic function of "chemotherapy-radiotherapy-immunotherapy";

[0029] (2) Most of the compounds claimed in the present invention showed good in vitro biological activity, among which the target polypeptide-coupled compound XXII had the best selectivity, with IC values ​​of XXII against liver cancer cells Hep3B and Huh-7. 50 The values ​​were 12.20 and 1.91 nM, respectively, and the IC values ​​for HeLa and LX-2 cells negative for GPC3 expression were 50 The values ​​were all greater than 100 nM;

[0030] (3) The present invention labels the polypeptide conjugate compound XXII with Cy5.5. The results of in vitro cell experiments show that the conjugate XXII can specifically bind to liver cancer cells and be efficiently internalized, thereby achieving targeted release of MMAE.

[0031] (4) The present invention has demonstrated through mechanism experiments that the polypeptide-coupled compound XXII significantly induces liver cancer cell cycle arrest in the G2 / M phase, which is the most sensitive to radiotherapy. When combined with low-dose (2Gy) radiotherapy, it can increase the formation of γH2AX foci, activate DNA damage repair, block cell progression, and reduce the survival rate of clone formation, thereby achieving a radiosensitization effect.

[0032] (5) When the polypeptide-coupled compound XXII claimed in the present invention is used in combination with low-dose radiotherapy, the polypeptide-coupled compound XXII combined with 2Gy radiotherapy shows a better in vivo tumor control effect than single therapy, highlighting its application potential in precise radiotherapy and chemotherapy of liver cancer. In addition, the polypeptide-coupled compound XXII can specifically induce immunogenic cell death in liver cancer cells, suggesting that it has anti-liver cancer immune activation potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Other features, objects and advantages of the present invention will become more apparent upon reading the detailed description of non-limiting embodiments with reference to the following drawings:

[0034] Figure 1 This is a difference diagram of the GPC3 protein expressed by Western Blot method in Example 10 of the present invention;

[0035] Figure 2 This is a difference diagram of GPC3 protein expressed by immunofluorescence method in Example 10 of the present invention;

[0036] Figure 3This is a difference diagram of GPC3 protein expression using flow cytometry in Example 10 of the present invention;

[0037] Figure 4 This is a bar graph showing the differences in GPC3 protein expression using flow cytometry strains in Example 10 of the present invention;

[0038] Figure 5 This is an immunofluorescence image of the polypeptide-coupled compound Cy5.5-XXII in Example 11 of the present invention;

[0039] Figure 6 This is the immunofluorescence image of naked peptide competition in Example 11 of the present invention;

[0040] Figure 7 This is an immunofluorescence experiment diagram of the internalization of the polypeptide-coupled compound Cy5.5-XXII in living Huh-7 cells in Example 11 of the present invention;

[0041] Figure 8 This is a graph showing the stability test results of the polypeptide-coupled compound XXII in Example 12 of the present invention in PBS, human serum, and mouse serum;

[0042] Figure 9 This is the specific release result in the cathepsin B system in Example 12 of the present invention;

[0043] Figure 10 This is a graph showing the effects of different concentrations of the polypeptide-coupled compound XXII on the cell cycles of Hep3B, Huh-7, and LX-2 cells according to Example 13 of the present invention;

[0044] Figure 11 This is a graph showing the effect of 50 nM of the polypeptide-coupled compound XXII on the cell cycle of liver cancer cells Huh-7 at different action times in Example 13 of the present invention;

[0045] Figure 12 This is a crystal violet staining image of the liver cancer cell Huh-7 on the 14th day after being irradiated with 2 Gy ionizing radiation, incubated with 10 nM polypeptide-coupled compound XXII, and their combined treatment in Example 14 of the present invention;

[0046] Figure 13 This is a diagram showing the quantitative analysis results of the colony formation experiment in Example 14 of the present invention;

[0047] Figure 14 This is a fluorescence image of double-stranded DNA breaks in liver cancer cell Huh-7 in Example 15 of the present invention;

[0048] Figure 15 This is a fluorescence image of γH2AX focus formation in liver cancer cell Huh-7 in Example 15 of the present invention;

[0049] Figure 16 This is a graph showing that pretreatment with the polypeptide-coupled compound XXII in Example 15 of the present invention can significantly enhance radiotherapy-induced γH2AX phosphorylation;

[0050] Figure 17 This is a diagram showing the phosphorylation of the DNA damage response protein CHK2 activated by pretreatment with the polypeptide-coupled compound XXII in Example 15 of the present invention;

[0051] Figure 18 This is a graph showing the CDK1 level after pretreatment with the polypeptide-coupled compound XXII in Example 15 of the present invention;

[0052] Figure 19 This is the immunofluorescence image of calreticulin in liver cancer cells in Example 16 of the present invention;

[0053] Figure 20 This is an immunofluorescence image of high mobility group protein B1 of liver cancer cells in Example 16 of the present invention;

[0054] Figure 21 This is a schematic diagram of the study in the mouse liver cancer Huh-7 transplanted tumor model in Example 17 of the present invention;

[0055] Figure 22 This is a graph showing the growth curve of mouse tumors in Example 17 of the present invention;

[0056] Figure 23 This is a graph showing the tumor inhibition rate of mice in Example 17 of the present invention;

[0057] Figure 24 This is a graph showing changes in mouse body weight in Example 17 of the present invention;

[0058] Figure 25 This is an H&E tissue staining image of mice in Example 17 of the present invention;

[0059] Figure 26 This is an immunohistochemical staining image of γH2AX of mouse tumors in Example 17 of the present invention. DETAILED DESCRIPTION

[0060] The present invention will be further described in detail below with reference to the embodiments, but the embodiments of the present invention are not limited thereto. Without departing from the above-mentioned technical concept of the present invention, various substitutions and changes can be made according to common technical knowledge and customary means in the field, and all of these should be included within the scope of the present invention.

[0061] The present invention provides a polypeptide coupling compound targeting GPC3, the general formula of which is as follows:

[0062] X——Linker——Y

[0063] Wherein, X is any polypeptide capable of targeting GPC3, including but not limited to the following polypeptide sequences: YFLTTRQ, RLNVGGTYFLTTRQ, ALLANHEELFQT, THVSPNQGGLPS, DYEMHLWWGTEL, DHLASLWWGTEL, and SNDRPPNILQKR.

[0064] Y is a drug with chemotherapy, radiotherapy and immunotherapy functions; specifically, it is MMAE, a drug with "chemotherapy-radiotherapy-immunotherapy" functions.

[0065] Linkers are any type of linker designed to couple the GPC3-targeting peptide to the drug MMAE. These include, but are not limited to, the common Val-Cit-PABC linker, which is specifically cleaved by cathepsin B. To achieve covalent attachment of the GPC3-targeting peptide to the linker, a maleimide acetyl group is introduced at the front of the linker, and a cysteine ​​residue is introduced at the N-terminus of the targeting peptide, enabling site-specific, efficient covalent coupling via a maleimide-thiol click chemistry reaction.

[0066] The specific preparation method of the above-mentioned polypeptide-coupled compound targeting GPC3 comprises the following steps:

[0067] Step S1: Preparation of intermediate MC-Val-Cit-PABC-MMAE;

[0068] Step S2: using a standard fluorenylmethyloxycarbonyl (Fmoc) solid phase peptide synthesis (SPPS) method to prepare polypeptides IX to XV;

[0069] Step S3: Preparation of GPC3-targeted polypeptide-coupled compounds, namely target polypeptide-coupled compounds XVI to XXII

[0070] Example 1:

[0071] This example provides a specific preparation process of the intermediate MC-Val-Cit-PABC-MMAE:

[0072] The specific preparation process of compound I is as follows: 4-aminobenzyl alcohol (2.46 g, 20.00 mmol) was dissolved in 20 mL of DMF, and then Fmoc-L-citrulline (7.95 g, 20.00 mmol), HATU (11.41 g, 30.00 mmol) and DIPEA (6.97 g, 40.00 mmol) were added to the solution. The mixture was stirred at room temperature for 3 hours and then concentrated under reduced pressure to obtain crude product 1. The product was purified by preparative reverse phase chromatography (water / acetonitrile system, Biotage The crude product was purified by Bio C18 chromatography column (particle size 20 μm, 80 g, eluted with acetonitrile gradient 10%-100% over 40 minutes) to obtain 8.04 g of white powder with a yield of 80%. 1 H NMR (400MHz, DMSO-d6) δ9.98 (s, 1H), 7.88 (ddd, J = 7.4, 2.1, 1.3Hz, 2H), 7.85–7.81 (m, 1H), 7. 74(dd,J=7.2,4.6Hz,1H),7.66(d,J=8.0Hz,1H),7.56(t,J=8.1Hz,2H),7.41(td,J=7.4,1.1H z,2H),7.37–7.29(m,2H),7.24(d,J=8.1Hz,2H),6.01(t,J=5.8Hz,1H),5.43(s,2H),5.13(s, 1H),4.43(s,2H),4.32–4.11(m,3H),3.13–2.87(m,2H),1.83–1.53(m,2H),1.51–1.36(m,2H); 13 C NMR(101MHz,DMSO-d6)δ174.52,171.8,159.30,143.03,139.88,137.96,137.88,137.78,129.42,12 7.78,127.41,121.86,120.51,119.35,110.25,63.08,55.53,32.87,27.1.HRMS(ESI-Q-TOF):calcd for C 28 H 31 N4O5 + [M+H] + ,503.2289;found,503.2500.

[0073] Compound II was prepared as follows: Compound I (7.54 g, 15.00 mmol) and diethylamine (10 mL) were dissolved in 20 mL of DMF and stirred at room temperature for 3 hours. The reaction solution was concentrated under reduced pressure, and the product was precipitated with ethyl acetate (5 × 20 mL). The resulting gel was collected by centrifugation, washed with methyl tert-butyl ether (3 × 20 mL), and dried to yield 4.26 g of a white powder (95% yield). 1H NMR (400MHz, DMSO-d6) δ7.57(d,J=8.4Hz,2H),7.23(d,J=8.4Hz,2H),6.02(t,J=5.3Hz,1H),5.40(s,2H),4.42 (s,2H),3.29(t,J=6.7Hz,1H),2.96(tt,J=12.4,6.0Hz,2H),1.60(dd,J=12.1,8.7Hz,1H),1.51–1.28(m,3H); 13 C NMR(101MHz,DMSO-d6)δ174.88,159.36,137.99,137.72,127.4,119.3,63.0,55.60,33.0,27.10.HRMS(ESI-Q-TOF):calcd for C 13 H 21 N4O3 + [M+H] + ,281.1608;found,281.1599.

[0074] The specific preparation process of compound III is as follows: Fmoc-L-valine (5.56 g, 14.00 mmol), HATU (7.99 g, 21.00 mmol) and DIPEA (4.88 mL, 28.00 mmol) were dissolved in 20 mL of DMF and mixed, and then compound II (3.92 g, 14.00 mmol) was added. The reaction solution was stirred at room temperature for 3 hours and then concentrated and purified by chromatography (water / acetonitrile system, Biotage Bio C18 chromatography column, particle size 20 μm, 80 g, acetonitrile gradient 10%-100% 40 minutes) to obtain compound 3 as a white powder (6.32 g, yield 75%). 1H NMR (400MHz, DMSO-d6) δ9.97(s,1H),8.10(d,J=7.5Hz,1H),7.88(d,J=7.5Hz,2H),7.73(t,J=8.0Hz,2H),7.53(d,J=8 .4Hz,2H),7.46–7.36(m,3H),7.32(td,J=7.5,1.0Hz,2H),7.23(d,J=8.5Hz,2H),5.98(t,J=5.7Hz,1H),5.41(s,2H),5 .13(t,J=5.7Hz,1H),4.42(d,J=5.7Hz,2H),4.42–4.37(m,1H),4.34–4.18(m,3H),3.92(dd,J=8.8,7.1Hz,1H),3.06–2 .91(m,2H),2.07–1.87(m,1H),1.73–1.64(m,1H),1.63–1.51(m,1H),1.46–1.32(m,2H),0.86(dd,J=10.9,6.8Hz,6H); 13 C NMR (101MHz, DMSO-d6) δ171.84,170.90,159.54,156.66,144.36,144.21,141.18,137.98,137.9,128.1,127.5,127. 46,125.8,120.55,119.42,66.2,63.11,60.64,53.59,47.1,30.94,30.0,27.24,19.7,18.7.HRMS(ESI-Q-TOF):calcd for C 33 H 40 N5O6 + [M+H] + ,602.2973;found,602.2969.

[0075] Compound IV was prepared as follows: Compound III (6.02 g, 10.00 mmol) and diethylamine (10 mL) were dissolved in 20 mL of DMF and stirred at room temperature for 3 hours. The reaction solution was concentrated under reduced pressure, and the product was precipitated with ethyl acetate (3 × 20 mL). The resulting gel was collected by centrifugation, washed with methyl tert-butyl ether (3 × 20 mL), and dried to yield 3.04 g of a white powder (80% yield). 1H NMR (400MHz, DMSO-d6) δ10.05(s,1H),8.14(d,J=5.8Hz,1H),7.54(d,J=8.5Hz,2H),7.2 3(d,J=8.5Hz,2H),6.03(t,J=5.7Hz,1H),5.43(s,2H),4.45(s,1H),4.43(s,2H),3.03( d,J=5.0Hz,1H),3.02–2.82(m,2H),2.00–1.86(m,1H),1.69(ddd,J=14.9,13.4,6.1Hz, 1H),1.63–1.51(m,1H),1.50–1.28(m,2H),0.88(d,J=6.8Hz,3H),0.78(d,J=6.8Hz,3H); 13 C NMR(101MHz,DMSO-d6)δ174.92,171.01,159.42,137.9,137.89,127.42,119.45,6 3.05,60.11,53.01,31.8,30.53,27.12,19.99,17.4.HRMS(ESI-Q-TOF):calcdfor C 18 H 30 N5O4 + [M+H] + ,380.2292;found,380.2291.

[0076] The specific preparation process of compound V is as follows: 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoic acid (1.48 g, 7.00 mmol), HATU (3.99 g, 10.5 mmol) and DIPEA (2.44 mL, 14.00 mmol) were dissolved in 15 mL of DMF and mixed, and then compound IV (2.66 g, 7.00 mmol) was added. The reaction solution was stirred at room temperature for 3 hours and then concentrated and purified by chromatography (water / acetonitrile system, Biotage Bio C18 chromatography column, particle size 20 μm, 80 g, acetonitrile gradient 10%-100% 40 minutes) to obtain compound 5 as a white powder (3.17 g, yield 79%). 1H NMR (400MHz, DMSO-d6) δ9.83(s,1H),7.99(d,J=7.6Hz,1H),7.75(d,J=7.6Hz,1H),7.47(d,J=8.4Hz,2H),7.16(d,J= 8.4Hz,2H),6.92(s,1H),5.92(t,J=5.7Hz,1H),5.35(s,2H),5.06(t,J=5.7Hz,1H),4.36(d,J=5.2Hz,2H),4.30(dd,J =13.6,8.2Hz,1H),4.15–4.05(m,1H),3.08(dd,J=12.8,6.7Hz,1H),3.01–2.80(m,2H),2.18–1.99(m,2H),1.96–1.80 (m,1H),1.62(dd,J=18.5,10.1Hz,1H),1.52(dd,J=9.1,4.5Hz,1H),1.48–1.05(m,8H),0.77(dd,J=12.1,6.8Hz,6H); 13 C NMR(101MHz,DMSO-d6)δ172.84,171.74,171.56,170.8,159.3,137.9,137.8,134.91,127.40,119.3,63.0 ,58.10,53.5,37.4,35.41,30.81,29.8,28.2,27.25,26.24,25.38,19.71,18.6.HRMS(ESI-Q-TOF):calcd for C 28 H 41 N6O7 + [M+H] + ,573.3031;found,573.3033.

[0077] Compound VI was prepared as follows: Compound V (2.86 g, 5.00 mmol), bis(4-p-nitrophenyl) carbonate (4.56 g, 15.00 mmol), and DIPEA (1.74 mL, 10.00 mmol) were dissolved in 40 mL of DMF and stirred at room temperature overnight. The solvent was removed under reduced pressure, and the product was precipitated with ethyl acetate (150 mL). The precipitate was collected by suction filtration, washed several times with ethyl acetate, and dried to yield 2.58 g of a brown amorphous powder in a 70% yield. 1H NMR (400MHz, DMSO-d6) δ10.08(s,1H),8.32(d,J=9.2Hz,2H),8.12(d,J=7.4Hz,1H),7.83(d,J=8.6Hz,1H),7.66(d,J=8.5Hz,2H) ,7.57(d,J=9.2Hz,2H),7.42(d,J=8.6Hz,2H),6.99(s,2H),6.02(t,J=5.6Hz,1H),5.44(s,2H),5.25(s,2H),4.39(dd,J=13.3,7 .9Hz,1H),4.24–4.11(m,1H),3.37(t,J=7.0Hz,2H),3.18(d,J=5.2Hz,1H),3.00(dtt,J=25.8,12.9,6.5Hz,2H),2.25–2.07(m,2 H),2.06–1.90(m,1H),1.72(dt,J=13.2,6.2Hz,1H),1.60(ddd,J=22.0,10.9,6.6Hz,1H),1.55–1.33(m,6H),1.29–1.13(m,6H); 13 C NMR (101MHz, DMSO-d6) δ172.87,171.82,171.55,171.22,159.44,155.7,152.4,145.63,134.89,129.96,129.77,126.67,125.87,123. 09,119.52,116.28,70.72,58.10,53.64,37.47,35.4,30.82,29.67,28.2,27.26,26.24,25.37,19.70,18.66.HRMS(ESI-Q-TOF):calcd for C 35 H 44 N7O 11 + [M+H] + ,738.3093;found,738.3093.

[0078] The specific preparation process of the intermediate MC-Val-Cit-PABC-MMAE is as follows: Compound VI (0.74 g, 1.00 mmol), MMAE (0.65 g, 0.90 mmol), HOBt (27 mg, 0.20 mmol) and pyridine (2 mL) were dissolved in 20 mL of DMF and stirred at room temperature overnight. The reaction solution was poured into ethyl acetate (100 mL) to precipitate, which was collected by suction filtration and washed with ethyl acetate (3×40 mL). The product was purified by preparative reverse phase chromatography (water / acetonitrile system, Biotage The product was separated and purified by Bio C18 column (particle size 20 μm, 80 g, 10%-100% acetonitrile gradient elution over 40 minutes) to obtain the intermediate MC-VCit-PABC-MMAE (533 mg) as a white amorphous powder with a yield of 45%. 1 H NMR (400MHz, DMSO-d6): δ9.95(d,J=6.9Hz,1H),8.09(d,J=7.6Hz,1H),7.80(d,J=8.6Hz 1H),7.59(d,J=8.3Hz,2H),7.34–7.26(m,6H),7.17(t,J=7.7Hz,1H),6.98(s,2 H),5.98(t,J=5.6Hz,1H),5.44–5.35(m,3H),5.08–4.96(m,2H),4.49(t,J=5.3 Hz,1H),4.42(d,J=5.7Hz,1H),4.41–4.32(m,1H),4.25(t,J=11.3Hz,1H),4.21 –4.14(m,1H),4.04–3.92(m,2H),3.77(d,J=9.8Hz,1H),3.60–3.53(m,1H),3.56 –3.35(m,5H),3.17–3.24(m,9H),3.12(s,2H),3.06–2.92(m,4H),2.86(d,J=14 .6Hz,3H),2.40(d,J=15.9Hz,1H),2.33–2.22(m,1H),2.22–2.07(m,4H),2.04– 1.901(m,3H),1.54–1.41(m,7H),1.24–1.22(m,5H),1.22–1.13(m,2H),1.10(t ,J=7.0Hz,1H),1.07–0.96(m,7H),0.92–0.72(m,26H).HRMS(ESI-Q-TOF):calcd for C 68 H 106 N 11 O 15 + [M+H] + ,1316.7864;found,1316.7889.

[0079] Example 2:

[0080] This example provides the specific process of solid phase synthesis, purification and structural identification of polypeptides IX to XV:

[0081] Peptide Synthesis: Peptides IX-XV were synthesized using a standard Fmoc solid-phase synthesis method on a Biotage Initiator+Alstra microwave synthesizer using Rink amide MBHA resin (0.358 mmol / g loading; Xi'an Lanxiao Technology LXSS09-1-1201) as a solid-phase support at a 0.3 mmol reaction scale. The specific process is as follows: Swelling: The resin was swollen in a 1:1 DCM / DMF mixture at room temperature with shaking at 500 rpm for 1 hour. Fmoc Deprotection: 20% piperidine / DMF solution was added sequentially, stirred at room temperature for 3 minutes, then drained and washed with DMF; 20% piperidine / DMF solution was added again, stirred at room temperature for 10 minutes, and washed four times with DMF. Coupling reaction: Add a DMF mixture (15 mL) of Fmoc-protected amino acid (5 equivalents, 0.05 M DMF solution), HATU (5 equivalents), HOBt (5 equivalents) and DIPEA (10 equivalents) to the deprotected resin, react under microwave irradiation at 75°C for 5 minutes, and wash with DMF four times. Cutting and purification: After sequence assembly is completed, the resin is manually washed three times with DCM and reacted with TFA / TIPS / H2O (95:2.5:2.5) cutting solution at room temperature for 3 hours. Remove TFA with nitrogen gas, precipitate the peptide with pre-cooled methyl tert-butyl ether (-20°C), centrifuge at 10,000 rpm for 5 minutes, discard the supernatant and vacuum dry the peptide precipitate. Note: All solvent volumes are 15 mL / time, and the operating temperature is room temperature where not marked.

[0082] Peptide purification: The crude peptide was purified by reverse phase high performance liquid chromatography (HPLC). A Shimadzu LC-20AP system was used, equipped with a dual-channel UV detector (detection wavelengths of 214 nm and 254 nm). The crude peptide was dissolved in an appropriate amount of DMSO, diluted with acetonitrile / water (1:1), and passed through an Ultimate 5μm XB-C18 preparative column ( Separation was performed using a flow rate of 8 mL / min. Mobile phases: Phase A was 0.1% TFA in water, and Phase B was 0.1% TFA in acetonitrile. Gradient program: Initially 10% B for 10 minutes, then linearly increased to 60% B over 40 minutes, then to 90% B over 10 minutes and held for 10 minutes.

[0083] Peptide Structural Characterization: The structures of the synthesized peptides IX to XV were confirmed by liquid chromatography-mass spectrometry (LC / MS). An Agilent 1200-6120 Q-TOF electrospray ionization high-resolution mass spectrometer was used with an ion source temperature of 350°C and a fragmentor voltage of 250 V, coupled with a Kinetex 2.6μm EVO-C18 column (50mm×3.0mm, Detection was performed using a flow rate of 1.2 mL / min. Mobile phases A and B were 0.1% TFA in water and 0.1% TFA in acetonitrile (LC / MS grade, purchased from Fisher Scientific). Gradient program: 15% B to 50% B over 1 minute, then to 90% B over 3 minutes and held for 2 minutes.

[0084] Peptide IX is a white powder, HRMS (ESI-Q-TOF): calculated for C 46 H 72 N 13 O 12 S + [M+H] + ,1030.5139; found,1030.4885.[M+2H] + ,found,515.7484.

[0085] Peptide X is a white powder. HRMS (ESI-Q-TOF): calculated for C 75 H 123 N 24 O 21 S + [M+H] + ,1727.9010; found,1727.9003.[M+2H] 2+ ,found,864.2005.

[0086] Peptide XI is a white powder, HRMS (ESI-Q-TOF): calculated for C 53 H 87 N 18 O 18 S + [M+H] + ,1295.6161; found,1295.6169.[M+2H] 2+ ,found,648.3113.

[0087] Peptide XII is a white powder, HRMS (ESI-Q-TOF): calculated for C 65 H 103 N 18 O 20 S + [M+H] + ,1487.7311; found,1487.7302.[M+2H] 2+ ,found,744.3701.

[0088] Peptide XIII is a white powder, HRMS (ESI-Q-TOF): calculated for C 70 H 101 N 18 O 19 S + [M+H] + ,1529.7206; found,1529.7184.[M+2H] 2+ ,found,765.3613.

[0089] Peptide XIV is a white powder. HRMS (ESI-Q-TOF): calculated for C 77 H 105 N 18 O 21 S2 + [M+H] + ,1681.7138; found,1681.7195.[M+2H] 2+ ,found,841.3605.

[0090] Peptide XV is a white powder. HRMS (ESI-Q-TOF): calculated for C 63 H 111 N 24 O 19 S + [M+H] + ,1539.8173; found,1539.8201.[M+2H] 2+ ,found,770.4132.

[0091] Example 3:

[0092] This example provides a specific preparation process of the polypeptide-coupled compound XVI, the structural formula of which is as follows:

[0093]

[0094] Peptide IX (51.51 mg, 0.05 mmol) was dissolved in DMF (6 mL), and MC-VCit-PABC-MMAE (68.83 mg, 0.05 mmol) and triethylamine (250 μL) were added. The mixture was stirred at room temperature under nitrogen for 5 hours. The crude product was purified by preparative HPLC (Shimadzu LC-20AP system, Ultimate 5 μm XB-C18 preparative column, mobile phase A: 0.1% TFA aqueous solution, B: 0.1% TFA acetonitrile solution; flow rate 8 mL / min; gradient: initial 10% B for 10 minutes, increased to 60% B within 40 minutes, then increased to 90% B and maintained for 10 minutes) to obtain peptide conjugated compound XVI (76 mg, yield 65%) as a white amorphous powder. HRMS (ESI-Q-TOF): calculated for C 114 H 176 N 24 O 28 S + [M+H] + ,2347.2770; found,2347.2800.[M+2H] 2+ ,found,1174.1410;[M+3H] 3+ ,found,783.0963. HPLC retention time: 14.728 minutes, purity: 97.96%. Mobile phases A: 0.1% TFA in water, B: 0.1% TFA in acetonitrile. Gradient: Initially 10% B for 3 minutes, linearly increasing to 60% B over 12 minutes and holding for 5 minutes; then increasing to 90% B over 3 minutes and holding for 2 minutes; flow rate: 1.0 mL / min.

[0095] Example 4:

[0096] This example provides a specific preparation process of the polypeptide-coupled compound XVII, the structural formula of the polypeptide-coupled compound XVII is as follows:

[0097]

[0098] By a similar method as in Example 3, polypeptide X was coupled with MC-VCit-PABC-MMAE and purified to obtain polypeptide conjugated drug XVII (yield 60%) as a white amorphous powder.

[0099] HRMS(ESI-Q-TOF):calcd for C 143 H 229 N 35 O 36 S 2+ [M+2H] 2+,1522.3437,found,1522.3169;[M+3H] 3+ ,found,1015.2138;[M+4H] 4+ , found, 761.6624. Retention time: 14.239 minutes, HPLC purity: 99.22%. Mobile phases A: 0.1% TFA in water, B: 0.1% TFA in acetonitrile. Gradient: Initially 10% B for 3 minutes, then linearly increase to 60% B over 12 minutes and hold for 5 minutes; then increase to 90% B over 3 minutes and hold for 2 minutes; flow rate: 1.0 mL / min.

[0100] Example 5:

[0101] This example provides a specific preparation process of the polypeptide-coupled compound XVIII, the structural formula of which is as follows:

[0102]

[0103] By a similar method as in Example 3, peptide XI was coupled with MC-VCit-PABC-MMAE and purified to obtain peptide conjugated drug XVIII (yield 50%) as a white amorphous powder. HRMS (ESI-Q-TOF): calculated for C 121 H 191 N 28 O 34 S + [M+H] + ,2612.3793; found,2612.3545.[M+2H] 2+ ,found,1306.6787;[M+3H] 3+ , found, 871.4542. Retention time: 13.883 minutes. HPLC purity: 99.06%. Mobile phases A: 0.1% TFA in water, B: 0.1% TFA in acetonitrile. Gradient: Initially 10% B for 3 minutes, then linearly increase to 60% B over 12 minutes and hold for 5 minutes; then increase to 90% B over 3 minutes and hold for 2 minutes. Flow rate: 1.0 mL / min.

[0104] Example 6:

[0105] This example provides a specific preparation process of the polypeptide-coupled compound XIX, the structural formula of which is as follows:

[0106]

[0107] By a similar method as in Example 3, peptide XII was coupled with MC-VCit-PABC-MMAE to obtain peptide conjugated drug XIX (yield 62%) as a white amorphous powder. HRMS (ESI-Q-TOF): calculated for C 133 H 207 N 28 O 36 S + [M+H] + ,2804.4943; found,2804.4766.[M+2H] 2+ ,found,1402.7410;[M+3H] 3+ , found, 935.4958. Retention time: 14.751 minutes. HPLC purity: 97.63%. Mobile phases A: 0.1% TFA in water, B: 0.1% TFA in acetonitrile. Gradient: Initially 10% B for 3 minutes, then linearly increase to 60% B over 12 minutes and hold for 5 minutes; then increase to 90% B over 3 minutes and hold for 2 minutes. Flow rate: 1.0 mL / min.

[0108] Example 7:

[0109] This example provides a specific preparation process of a polypeptide-coupled compound XX, the structural formula of which is as follows:

[0110]

[0111] By a similar method as in Example 3, peptide XIII was coupled with MC-VCit-PABC-MMAE and purified to obtain peptide conjugated drug XX (yield 45%) as a white amorphous powder. HRMS (ESI-Q-TOF): calculated for C 138 H 205 N 28 O 35 S + [M+H] + ,2846.4837; found,2846.4587.[M+2H] 2+ ,found,1423.7313;[M+3H] 3+ , found, 949.4899. Retention time: 15.685 minutes, HPLC purity: 97.58%. Mobile phases A: 0.1% TFA in water, B: 0.1% TFA in acetonitrile. Gradient: Initially 10% B for 3 minutes, then linearly increase to 60% B over 12 minutes and hold for 5 minutes; then increase to 90% B over 3 minutes and hold for 2 minutes; flow rate: 1.0 mL / min.

[0112] Example 8:

[0113] This example provides a specific preparation process of the polypeptide-coupled compound XXI, the structural formula of which is as follows:

[0114]

[0115] By a similar method as in Example 3, peptide XIV was coupled with MC-VCit-PABC-MMAE and purified to obtain peptide conjugated drug XXI (yield 49%) as a white amorphous powder. HRMS (ESI-Q-TOF): calculated for C 145 H 209 N 28 O 37 S 2+ [M+2H] 2+ ,1499.7385; found,1499.7345.[M+3H] 3+ , found, 1000.4928. Retention time: 15.619 minutes. HPLC purity: 97.66%. Mobile phases A: 0.1% TFA in water, B: 0.1% TFA in acetonitrile. Gradient: Initially 10% B for 3 minutes, then linearly increase to 60% B over 12 minutes and hold for 5 minutes; then increase to 90% B over 3 minutes and hold for 2 minutes. Flow rate: 1.0 mL / min.

[0116] Example 9:

[0117] This example provides a specific preparation process of the polypeptide-coupled compound XXII, the structural formula of the polypeptide-coupled compound XXII is as follows:

[0118]

[0119] By a similar method as in Example 3, peptide XV was coupled with MC-VCit-PABC-MMAE and purified to obtain peptide conjugated drug XXII (yield 59%) as a white amorphous powder. HRMS (ESI-Q-TOF): calculated for C 131 H 217 N 35 O 34 S 2+ [M+2H] 2+ ,1428.3019; found,1428.8000.[M+3H] 3+ ,found,952.8693.[M+4H] 4+, found, 714.6553. Retention time: 13.596 minutes. HPLC purity: 95.96%. Mobile phases A: 0.1% TFA in water, B: 0.1% TFA in acetonitrile. Gradient: Initially 10% B for 3 minutes, then linearly increase to 60% B over 12 minutes and hold for 5 minutes; then increase to 90% B over 3 minutes and hold for 2 minutes. Flow rate: 1.0 mL / min.

[0120] Example 10:

[0121] In this example, the above-prepared peptide conjugate drugs XVI-XXII were tested for their in vitro anti-tumor activity on different cell lines (IC 50 nM) experiments.

[0122] First, the expression levels of GPC3 protein in different cell lines were confirmed (Western Blot, immunofluorescence, and flow cytometry).

[0123] Western blot was used to analyze the expression level of GPC3 protein in Hep3B, Huh-7, LX-2 and HeLa cells. Specific steps: The cells were lysed with RIPA lysis buffer (product number P0013B, Beyotime) to extract total protein, and equal amounts of protein (50 μg) were separated by gel electrophoresis and transferred to PVDF membranes. The primary antibodies used were rabbit anti-GPC3 antibody (clone number SP86, Abcam) and mouse β-actin antibody (product number 66009-1-Ig, Proteintech), with a dilution ratio of 1:1000; the secondary antibodies were HRP-labeled goat anti-rabbit (product number RGAR001, Proteintech) and goat anti-mouse (product number RGAM001, Proteintech) antibodies, with a dilution ratio of 1:5000. After incubation and washing, enhanced chemiluminescence (ECL) was used for color development, and images were collected using the ChemiDocTM MP imaging system (BioRAD, USA). The experimental results are shown in Figure 2. Figure 1 As shown, GPC3 was highly expressed in Hep3B and Huh-7 cells, but not in HeLa and LX-2 cells.

[0124] The expression of GPC3 in Hep3B, Huh-7, LX-2 and HeLa cells was analyzed by immunofluorescence staining (CLSM) and flow cytometry (FCM). 5The cells were seeded at a density of 100 / dish in confocal culture dishes (catalog number BS-20-GJM, Biosun) and cultured overnight at 37°C and 5% CO2. The culture medium was discarded, and after washing three times with PBS, complete culture medium containing rabbit anti-GPC3 primary antibody (SP86, Abcam, 1:200 dilution) was added and incubated at 37°C for 4 hours; washed with PBS, fixed with 100% pre-cooled methanol for 10 minutes, and then Cy3-labeled goat anti-rabbit secondary antibody (catalog number A0516, Beyotime, 1:200 dilution) was added and incubated at 37°C in the dark for 1 hour; after washing with PBS, DAPI (catalog number C1005, Beyotime) was added to stain the nucleus. Images were collected using an Olympus SpinSR super-resolution confocal microscope. Flow cytometry analysis: After trypsinization and centrifugation, the cells were resuspended in pre-cooled PBS and analyzed by BD LSRFortessa TM The expression level of GPC3 was detected by flow cytometry. The images collected by confocal microscopy (CLSM) were as follows: Figure 2 As shown in Figure 2, Hep3B and Huh-7 cells showed significant fluorescence signals, while HeLa and LX-2 cells showed weak fluorescence. Figure 3 , Figure 4 As shown in Figure 3, GPC3 expression levels in human HCC cells (Hep3B and Huh-7) were significantly increased compared to normal hepatocytes (LX-2). These results confirm that GPC3 is specifically overexpressed in HCC cells.

[0125] Then, the MTT method was used to evaluate the in vitro antitumor activity of the peptide-coupled drugs XVI-XXII prepared in the present invention on Hep3B, Huh-7, LX-2 and HeLa cells. 3 The cells were seeded in a 96-well plate at a density of 100 μL / well (200 μL culture medium per well) and cultured overnight in a 37°C, CO2 incubator. The culture medium was discarded, and compound solutions of different concentrations were added, and the culture was continued for 72 hours at 37°C and 5% CO2. 20 μL of MTT solution (5 mg / mL, Beyotime, ST1537) was added to each well, and the supernatant was discarded after incubation for 4 hours. The precipitate was dissolved with DMSO. The absorbance (OD value) was measured at a wavelength of 570 nm using a microplate reader, and the concentration-effect curve was drawn using GraphPad Prism5.0 software (GraphPad Software) to calculate the half-maximal inhibitory concentration (IC) of the compound on the cells. 50 nM).

[0126] Finally, the experimental results are shown in Table 1.

[0127] Table 1 In vitro antiproliferative activity of compounds (IC 50 ,nM)

[0128]

[0129] The results of in vitro activity tests are shown in Table 1. Free MMAE showed a broad-spectrum inhibitory effect on all cell lines (IC 50 The activity of peptide-coupled compounds XVI, XVIII, XIX, XX, and XXI on Hep3B cells was weak (IC 50 >100nM; although peptide-coupled compound XVII showed significant inhibitory activity against Hep3B (81.23±18.23nM) and Huh-7 (67.90±24.72nM) cells, it lacked selectivity (HeLa: 70.41±20.35nM; LX-2: 30.82±11.23nM). Notably, peptide-coupled compound XXII exhibited the best selectivity: its IC against Hep3B (12.20±6.10nM vs 8.63±2.51nM) and Huh-7 (1.92±0.71nM vs 1.05±0.52nM) was 50 The cytotoxicity is close to that of free MMAE, but the cytotoxicity to GPC3 negative cells is very low (IC 50 The results showed that the peptide-coupled compound XXII could specifically kill GPC3-positive tumor cells and had higher safety than free MMAE.

[0130] Example 11:

[0131] This example uses a Cy5.5-labeled peptide conjugated compound XXII to test its binding and internalization in different cell lines.

[0132] The experimental process is as follows:

[0133] First, a peptide coupling compound XXII labeled with Cy5.5 was used to synthesize the peptide coupling compound Cy5.5-XXII. The structural formula of the peptide coupling compound Cy5.5-XXII is as follows:

[0134]

[0135] The specific preparation process is as follows: the peptide coupling compound XXII (14.27 mg, 0.005 mmol) was dissolved in DMF (1 mL), Cy5.5-NHS (3.58 mg, 0.005 mmol) and DIEA (30 μL) were added, and the mixture was stirred at room temperature for 3 hours under nitrogen protection. The crude product was purified by preparative HPLC (Shimadzu LC-20AP system, Ultimate 5 μm XB-C18 preparative column, mobile phase A: 0.1% TFA aqueous solution, B: 0.1% TFA acetonitrile solution; flow rate 8 mL / min; gradient: initial 10% B for 10 minutes, increased to 60% B within 40 minutes, then increased to 90% B and maintained for 10 minutes) to obtain the peptide coupling compound Cy5.5-XXII (9.23 mg, yield 54%) as a light blue amorphous powder. HRMS (ESI-Q-TOF): calculated for C 114 H 176 N 24 O 28 S + [M+2H] 2+ ,1711.4589; found,1711.4249.[M+3H] 3+ ,found,1140.9510;[M+4H] 4+ , found, 856.2159. Retention time: 17.420 minutes, HPLC purity: 98.60%. Mobile phases A: 0.1% TFA in water, B: 0.1% TFA in acetonitrile. Gradient: Initially 10% B for 3 minutes, then linearly increase to 60% B over 12 minutes and hold for 5 minutes; then increase to 90% B over 3 minutes and hold for 2 minutes; flow rate: 1.0 mL / min.

[0136] Secondly, confocal microscopy was used to investigate GPC3 receptor-mediated specific binding and internalization of the peptide-conjugated compound Cy5.5-XXII when incubated with different cell lines. Cells were seeded in confocal microscopy-specific glass-bottomed culture dishes (Boaosen Biotechnology) and incubated at 37°C, 5% CO2 for 24 hours. 10 μM Cy5.5-labeled peptide-conjugated compound Cy5.5-XXII was added and treated for 1 / 4 hour at 4°C or 37°C, followed by three washes with PBS. Cells were fixed with 100% pre-cooled methanol for 10 minutes and permeabilized with 0.5% Triton X-100 (Boaosen Biotechnology) for 10 minutes. After blocking with 5% BSA for 2 hours, rabbit anti-lysosomal associated membrane protein 1 (LAMP-1) polyclonal antibody (Cat. No. AF7353, Beyotime, 1:100 dilution) was added and incubated overnight at 4°C. Subsequently, FITC-conjugated goat anti-rabbit secondary antibody (Cat. No. A0562, Beyotime, 1:500 dilution) was added. After staining with antifade DAPI, images were acquired using an Olympus SpinSR super-resolution confocal microscope. For the naked peptide competition binding experiment, varying concentrations of peptide XV were added prior to incubation with the peptide-coupled compound Cy5.5-XXII.

[0137] Targeted binding Figure 5 As shown in the figure, the Cy5.5-labeled peptide conjugate compound Cy5.5-XXII exhibits strong red fluorescence signals in Hep3B and Huh-7 tumors, indicating that it specifically binds to GPC3 on the surface of the tumor cell membrane; while the fluorescence signals in GPC3-negative HeLa tumors and normal LX-2 cells are extremely weak, suggesting that the peptide conjugate compound XXII has high selectivity for HCC tumors and low affinity for normal non-liver cancer cells. In the competitive binding experiment, unlabeled peptide XV was added to compete with the peptide conjugate compound Cy5.5-XXII for binding to the GPC3 receptor on the surface of Huh-7 cells. The immunofluorescence imaging results are shown in the figure. Figure 6 As shown, the fluorescence intensity of Huh-7 cells decreased in a concentration-dependent manner with the competitor peptide XV. This result confirms that the specificity of the peptide-coupled compound Cy5.5-XXII binding is mediated by the targeting peptide, rather than the fluorescent group (Cy5.5) or toxin (MMAE).

[0138] Live cell confocal imaging was used to observe the internalization and operation of the peptide-coupled compound Cy5.5-XXII in Huh-7 cells. Figure 7As shown: After incubating Huh-7 cells at 37°C for 30 minutes, red fluorescent signals were distributed intracellularly. Co-staining with Lysotracker revealed significant colocalization of Cy5.5-22 (red) with lysosomes (green). This demonstrates that the conjugate achieves targeted delivery of MMAE and exerts specific anti-HCC activity through specific binding, internalization, and lysosomal accumulation of the GPC3 receptor.

[0139] Example 12:

[0140] In this example, the polypeptide-coupled compound XXII was used as the research object to verify its plasma stability and to conduct a specific release experiment under the action of cathepsin B.

[0141] The plasma stability of peptide conjugate compound XXII was tested using LC-MS / MS. Specifically, peptide conjugate compound XXII was dissolved in preheated 37°C human plasma, mouse plasma, or PBS (final concentration 500 μM). Samples were collected at 0, 0.25, 0.5, 1, 2, 4, 8, 24, and 48 hours. Methanol was added to terminate the enzymatic reaction and precipitate plasma proteins. The samples were centrifuged at 10,000 rpm for 10 minutes, and the supernatant was collected for LC-MS / MS analysis. LC-MS / MS was also used to investigate the specific cleavage of peptide conjugate compound XXII by cathepsin B. Specifically, cathepsin B (Cat. No. HY-P7993A, MCE) was activated for 15 minutes at room temperature in a buffer containing 25 mM sodium acetate, 1 mM EDTA, and 9.2 mM DTT (pH 5.5). After activation, the enzyme (final concentration 100 nM) was incubated with free MMAE or peptide-conjugated compound XXII (final concentration 20 μM) in reaction buffer (25 mM sodium acetate, 1 mM EDTA, pH 5.5) at 37°C. Samples were collected at 0, 5, 10, 20, 30, 40, 50, 60, and 90 minutes, and the reaction was immediately terminated by the addition of acetonitrile. The mixture was vortexed and allowed to stand at -20°C for 5 minutes. The samples were centrifuged at 10,000 rpm for 5 minutes, and the supernatant was collected for LC-MS / MS analysis. For inhibitor experiments, cathepsin B inhibitor (Cat. No. HY-103350, MCE) was pre-added to the reaction buffer prior to mixing with the substrate.

[0142] Plasma stability test results: Plasma stability is a key parameter in the development of peptide-coupled drugs. It is necessary to ensure that it remains intact in the circulation after intravenous injection until it targets the tumor. Peptide-coupled compound XXII (500 μM) was incubated with PBS, human plasma, and mouse plasma at 37°C for 48 hours, and the stability was monitored by LC-MS / MS. The results are as follows Figure 8As shown, the peptide-coupled compound XXII remained stable in PBS for 48 hours; after 8 hours in human plasma, 57.2% of the intact drug remained, and after 48 hours, it was 25.3% (half-life t 1 / 2 =8.8 hours); however, it degraded rapidly in mouse plasma, with only 19.9% ​​remaining after 8 hours (t 1 / 2 = 4 hours). The above results of the plasma stability experiment show that the peptide-coupled compound XXII has sufficient plasma stability and can be used for subsequent in vivo pharmacodynamic studies.

[0143] Results of cathepsin B specific release experiment: To verify whether the Val-Cit linker in peptide conjugate compound XXII can be specifically cleaved by cathepsin B to release MMAE, an enzyme cleavage experiment was performed in vitro under simulated lysosomal conditions (pH 5.5 buffer). The LC-MS / MS quantitative test results are as follows: Figure 9 As shown, the release of MMAE was time-dependent and nearly complete within 30 minutes; the release was blocked after the addition of cathepsin B inhibitor, confirming that cathepsin B can mediate the cleavage of the linker of the peptide-coupled compound XXII and precisely regulate the release of MMAE.

[0144] Example 13:

[0145] This example provides an experiment on the effects of polypeptide XV, MMAE, and polypeptide-conjugated compound XXII on the cell cycle of different cell lines (flow cytometry).

[0146] Flow cytometry was used to analyze the effect of different concentrations of peptide-coupled compound XXII on the cell cycle after incubation with Hep3B, Huh-7 and LX-2 cells for 24 hours. Specific operation: Hep3B, Huh-7 and LX-2 cells were incubated with DMSO and different concentrations of peptide-coupled compound XXII for 24 hours. After incubation, the cells were fixed with pre-cooled methanol and incubated at 4°C overnight, and then stained with PI / RNase staining solution (Cat. No. C1052, Beyotime). BD LSRFortessa TM Flow cytometric analysis was performed using a flow cytometer (BD Biosciences).

[0147] Effects of different concentrations of peptide-coupled compound XXII on the cell cycle of Hep3B, Huh-7 and LX-2 cells Figure 10The results showed that peptide-coupled compound XXII can dose-dependently induce cell cycle arrest in the G2 / M phase of GPC3-positive cells (Hep3B, Huh-7). For example, under the action of 10nM, 30nM, and 100nM peptide-coupled compound XXII, the proportion of Huh-7 cells in the G2 / M phase increased from 18.88% to 35.65%, 49.02%, and 76.34%, respectively; while the cell cycle distribution of GPC3-negative LX-2 cells was not significantly affected. The experimental results are consistent with its cytotoxicity characteristics (Table 1). In addition, Figure 11 As shown in the results, the G2 / M phase arrest induced by peptide-coupled compound XXII increased in a time-dependent manner. For example, when Huh-7 cells were treated with 50 nM peptide-coupled compound XXII for 18 hours, the proportion of cells in the G2 / M phase was 44.55%. When the incubation time was increased to 30 hours, it increased to 54.83%. At the same time, when the co-incubation time was 24 hours, the G2 / M phase cell cycle arrest reached its peak.

[0148] Example 14:

[0149] This example conducts an experiment to verify the radiosensitization effect of the polypeptide-coupled compound XXII on the liver cancer cell line Huh-7:

[0150] Given that peptide-conjugated compound XXII specifically arrests liver cancer cells in the G2 / M phase, the most radiosensitive phase, we conducted a colony formation assay to investigate its radiosensitizing effect on the Huh-7 liver cancer cell line. Specific procedures: 800 cells were seeded per well of a 6-well plate and incubated overnight with peptide-conjugated compound XXII. After 24 hours, the cells were removed from the plate and irradiated with 2 Gy of radiation. Fresh medium was then replaced and cultured for an additional 10-14 days. At the conclusion of the experiment, the cells were fixed with methanol, stained with crystal violet, and colonies were counted. All experiments were repeated in duplicate.

[0151] Based on the effect of peptide-coupled compound XXII on the Huh-7 cell cycle, Figure 18 As shown, Huh-7 cells were pretreated with 10 nM peptide-coupled compound XXII overnight and then irradiated with 2 Gy. On the 14th day after treatment, crystal violet staining showed the results as follows Figure 12 and Figure 13 As shown, compared with peptide-coupled compound XXII or radiotherapy alone (2Gy), the cell clones in the combined treatment group were significantly smaller. The results of this clone formation experiment indicate that peptide-coupled compound XXII can enhance the therapeutic effect of radiotherapy on HCC through synergistic effects.

[0152] Example 15:

[0153] This example verifies the effects of the polypeptide-coupled compound XXII on double-stranded DNA breaks, γH2AX focus formation, and activation of the DNA damage repair signaling pathway in liver cancer cell Huh-7.

[0154] Because radiotherapy can kill tumor cells by inducing DNA double-strand breaks (DSBs), γH2AX is a prominent molecular marker of DSBs. We used immunofluorescence to examine the DNA damage response of Huh-7 cells synergistically with peptide-conjugated compound XXII in response to low-dose radiotherapy. Huh-7 cells were starved of 1% FBS serum for 24 hours, seeded on slides, and treated overnight with peptide-conjugated compound XXII (100 nM). They were then irradiated with 2 Gy, washed with PBS 30 minutes later, fixed with 100% pre-cooled methanol for 10 minutes, and permeabilized with 0.5% Triton X-100 (Boosun Biotechnology) for 10 minutes. After blocking with 5% BSA for 2 hours, rabbit anti-γH2AX antibody (Cat. No. ET1602-2, Huaan Biotechnology, 1:1000 dilution) was added and incubated at 37°C for 1 hour. Cy3-conjugated goat anti-rabbit secondary antibody (Cat. No. A0516, Beyotime, 1:200 dilution) was then added. After nuclear staining with DAPI, images were acquired using a Nikon ECLIPSE Ti2-U fluorescence microscope. Western blot analysis was also performed to examine the effects of peptide-conjugated compound XXII in combination with low-dose radiotherapy on DNA damage repair signaling pathways in Huh-7 cells. Specifically, Huh-7 cells were pretreated with peptide-conjugated compound XXII (100 nM) for 24 hours before irradiation with 2 Gy. Cells were harvested 30 minutes after irradiation and lysed with RIPA lysis buffer (Cat. No. P0013B, Beyotime) containing protease / phosphatase inhibitors for protein extraction. Protein concentration was determined using the BCA assay (Cat. No. P0012S, Beyotime). Proteins were separated by 10% SDS-polyacrylamide gel electrophoresis and transferred to a PVDF membrane. The membranes were blocked with 5% skim milk for 2 hours at room temperature. Primary antibodies were added for overnight incubation at 4°C, followed by three 10-minute washes in TBST. The membranes were then incubated with an HRP-conjugated secondary antibody (Proteintech) for 2 hours at room temperature. BeyoECL Plus developer (Cat. No. P0018S, Beyotime) was used for color development, and images were acquired using the ChemiDoc™ MP imaging system (Bio RAD).

[0155] DNA double-strand break analysis results Figure 14 As shown in Figure 2, immunofluorescence staining was used to detect γH2AX focus formation. It was found that compared with the use of peptide-coupled compound XXII alone or radiotherapy (2Gy), the combination of peptide-coupled compound XXII and radiotherapy for 24 hours significantly increased the γH2AX fluorescence signal in Huh-7 cells. Quantification showed that the number of γH2AX foci in the combined group reached 18±2, which was significantly higher than that in the control group (6±2). Figure 15At the same time, Western blot confirmed that pretreatment with peptide-coupled compound XXII could significantly enhance radiotherapy-induced γH2AX phosphorylation, as shown in Figure 2. Figure 16 As shown, it shows that it improves radiotherapy sensitivity by increasing DNA damage. The study of the molecular mechanism of DNA damage repair showed that radiotherapy activates the phosphorylation of DNA damage response protein CHK2 (a marker of DSBs repair), and pretreatment with peptide-coupled compound XXII further enhances this effect. Figure 17 Phosphorylated CHK2 inhibits CDC25C activity, and the expression of CDC25C in the combined treatment group was significantly reduced, as shown in Figure 17 In addition, the combined treatment also downregulated CDK1 levels, as shown in Figure 18 As shown, it suggests that the peptide-coupled compound XXII activates the CHK2-CDC25C pathway, inhibits CDK1, blocks the cell G2 / M phase process, thereby enhancing the cumulative effect of radiotherapy on DNA damage and synergistically inhibiting tumor growth.

[0156] Example 16:

[0157] This example verifies that the polypeptide-coupled compound XXII can induce immunogenic cell death (ICD) in liver cancer cells Huh-7, and has the potential to immunize against liver cancer.

[0158] MMAE is known to promote the release of tumor antigens and modulate immune responses in the tumor microenvironment by inducing immunogenic cell death (ICD). To evaluate whether peptide-conjugated compound XXII induces ICD in HCC cells, we examined the membrane exposure of calreticulin (CALR) and the extracellular release of high-mobility group box 1 (HMGB1). Using immunofluorescence, we detected the extracellular exposure of calreticulin (CALR). Huh-7 cells were seeded in culture dishes overnight and treated with peptide-conjugated compound XXII (100 nM) for 24 hours. After thorough washing with PBS, the cells were fixed with 100% pre-cooled methanol for 10 minutes and permeabilized with 0.5% Triton X-100 (Boosun Biosciences) for 10 minutes. After blocking with 5% BSA for 2 hours, rabbit anti-CALR antibody (Cat. No. DF3139, Affinity, 1:300 dilution) was added and incubated at 37°C for 1 hour. Cy3-conjugated goat anti-rabbit secondary antibody (Cat. No. A0516, Beyotime, 1:200 dilution) was then added. Nuclear staining with DAPI was performed, and images were acquired using a Nikon ECLIPSE Ti2-U fluorescence microscope. Immunofluorescence was used to detect the nuclear release of high-mobility group box 1 (HMGB1). Huh-7 cells were seeded overnight in culture dishes and treated with peptide-conjugated compound XXII (100 nM) for 24 hours. After washing with PBS, the cells were fixed with 100% pre-cooled methanol for 10 minutes and permeabilized with 0.5% Triton X-100 for 10 minutes. After blocking with 5% BSA for 2 hours, rabbit anti-HMGB1 antibody (Cat. No. AF7020, Affinity, 1:300 dilution) was added and incubated at 37°C for 1 hour, followed by Cy3-conjugated goat anti-rabbit secondary antibody (Cat. No. A0516, Beyotime, 1:200 dilution). Nuclear staining with DAPI was performed, and images were acquired using a Nikon ECLIPSE Ti2-U fluorescence microscope.

[0159] After Huh-7 liver cancer cells were treated with peptide-coupled compound XXII (100 nM) for 24 hours, the immunofluorescence imaging results were as follows: Figure 19 As shown in the figure, peptide-coupled compound XXII can significantly induce CALR externalization, and the exposure of CALR on the cell membrane surface is significantly increased. At the same time, immunofluorescence analysis of HMGB1 showed that HMGB1 was mainly located in the cell nucleus before treatment with peptide-coupled compound XXII, and was transferred to the cytoplasm after treatment, as shown in the figure. Figure 20 The above results all confirm that the peptide-coupled compound XXII can effectively induce ICD in HCC cells and has anti-immune and anti-liver cancer potential.

[0160] Example 17:

[0161] In this example, the prepared polypeptide-coupled compound XXII was used to conduct an in vivo tumor inhibition experiment in synergy with low-dose (2 Gy) radiotherapy.

[0162] To evaluate whether the radiosensitizing effect of peptide-coupled compound XXII can enhance the response to radiotherapy in vivo, we further studied its anti-tumor effect in combination with low-dose radiotherapy. Specifically, in the in vivo precision chemoradiotherapy paradigm, female BALB / C nude mice were subcutaneously inoculated with Huh-7 cells (5×10 6 When the tumor reaches 100-200 mm 3 At the same time, the mice were randomly divided into a normal saline control group, a 22 (1 mg / kg) single-drug group, a single radiotherapy group (2 Gy), and a peptide-coupled compound XXII (1 mg / kg) + radiotherapy group (2 Gy) combined group. The peptide-coupled compound XXII (1 mg / kg) was injected intravenously once a week for 3 consecutive weeks, followed by 2 Gy radiotherapy for 2 consecutive days. During radiotherapy, only the tumors on the hind limbs of the mice were exposed, while other parts of the body were shielded with lead. The weight and tumor size of the mice were measured once every 2 or 3 days, and the formula for calculating the tumor volume was: tumor size = 1×w 2 = / 2 (l, long diameter; W, short diameter). Tumor size-time and body weight-time curves were plotted using GraphPad Prism 5.0. After treatment, mice were sacrificed, and tumor tissues were harvested for γH2AX immunohistochemical staining. Hearts, livers, spleens, lungs, and kidneys were also stained with H&E.

[0163] When the tumor volume reaches 100mm 3 When Huh-7 tumor-transplanted mice were treated with peptide-coupled compound XXII, they were given 2 Gy radiotherapy for two consecutive days per week for three weeks. Figure 21 The efficacy was evaluated on day 22 and it was found that although the dose of peptide-coupled compound XXII was only 1 mg / kg, the tumor volume (223 mm 3 ) were significantly lower than those in the blank control group (p<0.0001), the single-drug peptide-coupled compound XXII group (p<0.0001) and the single radiotherapy group (p<0.005). Figure 22 , Figure 23 As shown. Peptide-coupled compound XXII alone did not show significant tumor inhibition effect. No weight loss was observed during the treatment. Figure 24 As shown in Figure 2, H&E staining of major organs (heart, liver, spleen, lung, and kidney) showed that the combined treatment had no significant toxicity. Figure 25 Analysis of tumor samples on day 22 showed that the number of γH2AX-positive cells in the combined group was significantly higher than that in the radiotherapy or drug-only group. Figure 26 These results suggest that peptide-coupled compound XXII can synergize with radiotherapy through tumor-selective radiosensitization, providing a new strategy for precise chemoradiotherapy of hepatocellular carcinoma.

[0164] Although the embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the claims and their equivalents.

Claims

1. A polypeptide-coupled compound targeting GPC3, the general formula of which is as follows: in, X is any polypeptide capable of targeting GPC3; Y is a drug with chemotherapy, radiotherapy, and immunotherapy functions; Linker is a connector for coupling GPC3 targeting peptides with the drug MMAE. The front end of the linker introduces a maleimide acetyl end, and the N-terminus of the targeting peptide introduces a cysteine, and a maleimide-thiol click chemistry reaction is used for site-specific and efficient covalent coupling.

2. A polypeptide-coupled compound targeting GPC3 according to claim 1, characterized in that: The X is a polypeptide comprising the following amino acid sequence: YFLTTRQ, RLNVGGTYFLTTRQ, ALLANHEELFQT, THVSPNQGGLPS, DYEMHLWWGTEL, DHLASLWWGTEL, SNDRPPNILQKR.

3. A polypeptide-coupled compound targeting GPC3 according to claim 1 or 2, characterized in that: The Y is the drug MMAE.

4. A polypeptide-coupled compound targeting GPC3 according to claim 1 or 2, characterized in that: The linker includes a common Val-Cit-PABC type linker that can be specifically cleaved by cathepsin B.

5. A method for preparing a polypeptide-coupled compound targeting GPC3, characterized in that: The following steps are involved: Step S1: Preparation of intermediate MC-Val-Cit-PABC-MMAE; Step S2: Using the standard Fmoc solid-phase peptide synthesis SPPS method, Rink amide-MBHA resin was used as the starting material. An Fmoc-protected amino acid was added. In the presence of HATU, HOBt, and DIPEA, a GPC3-targeting peptide was synthesized on solid phase. A cysteine ​​residue was introduced at the N-terminus of the GPC3-targeting peptide to synthesize peptides IX to XV. Step S3: Peptides IX to XV were mixed with MC-Val-Cit-PABC-MMAE and Et3N, respectively, reacted at room temperature for 5 hours, and purified by preparative HPLC to obtain peptide-coupled compounds XVI to XXII.

6. The method for preparing a polypeptide-coupled compound targeting GPC3 according to claim 5, characterized in that: The specific process of preparing the intermediate MC-Val-Cit-PABC-MMAE in step S1 is as follows: Step S11: Using Fmoc-L-Cit-OH as a starting material, reacting with p-aminobenzyl alcohol under HATU and DIPEA catalysis to obtain compound I through amidation; Step S12: using compound I to remove Fmoc under the action of Et2NH to obtain compound II; Step S13: Compound II reacts with Fmoc-L-Val-OH under HATU and DIPEA catalysis to obtain compound III through amidation. Step S14: Compound III is subjected to Fmoc removal under the action of Et2NH to obtain compound IV; Step S15: Compound IV reacts with 6-maleimidocaproic acid under HATU and DIPEA catalysis to obtain compound V through amidation. Step S16: Compound V reacts with di(p-nitrobenzene) carbonate under DIPEA catalysis to obtain compound VI; Step S17: Compound VI is catalyzed by HOBt and pyridine to obtain the intermediate MC-Val-Cit-PABC-MMAE.

7. The method for preparing a polypeptide-coupled compound targeting GPC3 according to claim 5 or 6, characterized in that: The specific process of synthesizing polypeptides IX to XV in step S2 is as follows: Step S21: Using Rink amide-MBHA resin as a solid support, the Fmoc protecting group was deprotected with a 20% anhydrous piperidine solution in DMF. HATU, HOBt, and DIPEA were used as coupling agents. The deprotected resin was coupled with 5.0 equivalents of an Fmoc-protected amino acid in anhydrous DMF by microwave reaction at 75°C for 5 minutes. The order of amino acid addition corresponded to the order of the peptide sequence, ultimately yielding a peptide capable of targeting GPC3 and having an N-terminal cysteine ​​modification. Step S22: Add TFA / TIPS / H2O (v / v / v) in a ratio of 95:2.5:2.5 to cleave the polypeptide from the resin. The synthesized polypeptide is separated from the resin and then filtered. The resulting filtrate is poured into -20°C methyl tert-butyl ether to form a large amount of white precipitate, which is collected by centrifugation. Step S23: The precipitate is washed 2-3 times with cold methyl tert-butyl ether to obtain a crude peptide; Step S24: Separate and purify by preparative HPLC to obtain polypeptides IX to XV.

8. The method for preparing a polypeptide-coupled compound targeting GPC3 according to claim 5 or 6, characterized in that: In step S21, the ninhydrin method is used to confirm the completion of the coupling of each Fmoc-protected amino acid.

9. A drug for preventing, diagnosing, and / or treating diseases associated with GPC3 expression, comprising the GPC3-targeting polypeptide conjugate compound according to any one of claims 1 to 4 or the GPC3-targeting polypeptide conjugate compound prepared according to any one of claims 5 to 8 as a main component, and the remaining components being pharmaceutically acceptable, non-toxic, and non-inert pharmaceutical carriers and / or excipients.

10. The drug for preventing, diagnosing and / or treating diseases related to GPC3 expression according to claim 9, characterized in that: Prevention, diagnosis and / or treatment of diseases associated with GPC3 expression include liver cancer and tumors associated with GPC3 expression.

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